Scar formation on human skin post-wound closure results in physical and emotional damage to patients. Collagen is a key player in the wound healing process; however, excess production and deposition of collagen fibrils is a major cause of fibrosis, or scar formation. Thus, a wound treatment that utilizes the healing cues of collagen in a controlled manner while reducing collagen synthesis and deposition could improve scarless wound healing efforts. To this end, we propose to develop a Designer Collagen (DC) protein that binds to both cellular integrins known to contribute to wound healing and fibronectin (FN) in the wound bed. I hypothesize that wounds treated with DCs that interact with FN and collagen-based integrins ?1?1 and/or ?2?1 will heal at comparable or better rates than conventional collagen treatments and have reduced collagen synthesis and deposition to translate to a reduced visible scar. DCs are based upon a protein discovered from group A streptococcus (Scl2.28). These proteins form a stable triple helix similar to that of collagen, which is required for effective integrin binding; however, DCs serve as a biological blank slate, devoid of native collagen cues, that can then be modified with desired sites that impart selective bioactivity. A FN binding site will be incorporated into DCs that contain collagen-based binding sites for integrins ?1?1 and/or ?2?1, which are implicated in affecting fibrosis, or scarring, as well as promotig cell adhesion, proliferation, and migration throughout wound healing. This will allow for: 1) controlled integrin activation to promote the needed cell proliferation and migration for wound closure, 2) a physical anchor between DCs and the wound bed, and 3) a reduction in native collagen-FN interactions. The proposed work comprises two aims: (1) Utilize DCs with varied integrin ?1?1 and ?2?1 binding site affinity and avidity to elucidate the effects of integrin variables on cell signaling. A library of DCs with varied integrin ?1?1 and ?2?1 binding site affinity and avidity (number of binding sites) will be utilized to independently determine the effects of integrin variables on microvascular endothelial cell and dermal fibroblast signaling and phenotype (collagen synthesis, adhesion, spreading, migration, and proliferation). These proteins will also be applied to wounds in integrin ?1- and ?2-null mice to assess the effects of integrin variables in vivo. (2) Characterize DC-FNs with varied FN binding affinities to determine potential formulations that competitively bind with collagen. PCR insertion methods will be used to produce DC-FNs with varied FN binding affinities. The resulting protein structure, FN interactions, and integrin interactions will be characterized. DC-FN formulations that maintain the properties of their template DCs will be assessed in an in vitro 3D human skin equivalent wound healing model to measure wound closure rates and collagen synthesis. Then, an in vivo mouse excisional wound model will be used to determine the efficacy of DC-FNs at promoting healing, anchoring in the wound bed, and improving collagen deposition and organization. The results from these two aims will provide the needed tools to manufacture a DC-FN that has both the desired cellular signaling for promoting wound healing and strong FN interactions to anchor in the wound bed in place of collagen. Through this work, we seek to develop a technology that provides the healing capabilities of collagen while reducing scar formation as we increase fundamental understanding of integrin signaling and collagen-FN interactions in wound healing.